In the intact substrate, casein is heavily labeled with TAMRA, resulting in almost total quenching of the conjugate's fluorescence. Compared to the fluoresceinated casein, TAMRA-labeled casein demonstrates pH-insensitive fluorescence. It is readily used for continuous fluorometric measurement of protease activity. Protease-catalyzed hydrolysis relieves this quenching conjugate, yielding brightly red fluorescent dye-labeled peptides. The increase in fluorescence intensity is directly proportional to protease activity. The lightly TAMRA-labeled casein may be useful for a continuous assay if monitored by fluorescence polarization.